Arginine Deiminase Mutant with Improved Enzyme Activity and Temperature Stability and Application Thereof

ABSTRACT

An arginine deiminase mutant with improved enzyme activity and temperature stability and application thereof were provided, belonging to the technical field of genetic engineering and enzyme engineering. The arginine deiminase mutant is proline, namely Gly292 Pro, mutated from glycine near an enzyme active center. A wild-type arginine deiminase arcA coding gene is molecularly modified by a site-directed mutation technique to obtain a mutant enzyme ADIG292P, which has glycine at position 292 of an amino acid sequence of the wild type arginine deiminase mutated to proline. The arginine deiminase, modified by site-directed mutation, of the present invention has 1.5 times of increase in enzyme activity and 5.43 times of increase in half-life period at 40° C. compared with the wild-type enzyme, which solves the problems of low catalytic ability and temperature stability during the catalytic synthesis of citrulline using arginine deiminase, and lays a foundation for industrial production of efficient synthesis of citrulline and medication application.

TECHNICAL FIELD

The disclosure herein relates to the field of genetic engineering andenzyme engineering, which relates to an arginine deiminase mutant withimproved enzyme activity and temperature stability and applicationthereof,

BACKGROUND

Arginine deiminase (EC 3.5.3.6), abbreviated as ADI, can hydrolyzearginine to produce citrulline and ammonia. Since the first report in1933, the enzyme has been found in streptococcus lactiae, streptococcusfaecalis, yeast, pseudomonas, mycoplasma, halobacterium and someeukaryotic cells, etc. Arginine deaminase is wide in source, and hasbeen found in bacteria, archaebacteria and some eukaryotic cells. Thereare obvious differences in properties of ADI of different microbialsources, such as molecular weight range, optimal pH, optimaltemperature, etc. However, all the ADI has the ability to decomposearginine.

Citrulline, also called as carbamylornithine ornithin, gets the name asbeing extracted from watermelon initially. In recent years, it has beenfound by researches that citrulline has the physiological functions ofscavenging free radicals, affecting nitrogen balance in the human body,immune regulation of the cardiovascular system, and the like. Therefore,the application of citrulline in food and medicine aspects isincreasing. Because of the functions of aging resisting, immunityenhancement, athlete muscle strength and endurance improvement and thelike of citrulline, citrulline has been added into sports functionaldrinks produced by a Canadian company. At the same time, health skincare products containing citrulline are also sold on the market inEuropean and American countries.

Citrulline is mainly produced by four methods, i.e., a chemical method,an extraction method, a fermentation method and an enzymic method. Amongthem, the chemical method is currently the main production mode, butby-products such as copper ions and hydrogen sulfide produced during aproduction process have an adverse impact on the environment. Both theextraction method and the fermentation method for citrulline productionhave the disadvantages of low yield and high cost, and thus are notconducive to large-scale production. On the contrary, the enzymaticmethod for citrulline production has the advantages of mild reactionconditions, high conversion efficiency, simple extraction process andthe like, so the enzymatic method has high research and productionvalue. Therefore, the search for safe and stable arginine deiminase withhigh catalytic efficiency is the key issue for enzymatic citrullineproduction.

Sit-directed mutagenesis as a major means of molecular transformationrefers to a technology of introducing a specific base pair at adesignated site of a target DNA fragment to change an amino acidsequence encoded by the. Site-directed mutagenesis is more rapid, directand cost-effective than other strategies for improving a molecularstructure, so it is one of the most commonly used genetic modificationmeans in laboratories.

SUMMARY

The object of the invention is to improve the enzymatic activity and thetemperature stability of modified arginine deiminase by means ofmolecular modification on arginine deiminase and to use the modifiedarginine deiminase for citrulline production and medicine.

The invention provides an arginine deiminase mutant with site-directedmutation modification. The arginine deiminase mutant is obtained byapplying a site-directed mutagenesis technique based on an argininedeiminase gene of Enterococcus faecalis SK32.001. An amino acid sequenceof the arginine deiminase mutant is SEQ ID No. 1, and the argininedeiminase mutant is encoded by an nucleotide sequence of SEQ ID No. 2.

The amino acid mutation happens outside an arginine deiminase proteinstructure, and the mutation may increase the protein hydrophobicinteraction or electrostatic interaction.

Technical solutions of the invention: an arginine deiminase mutant withimproved enzyme activity and temperature stability, of which an aminoacid sequence is SEQ ID No.1, is provided.

A nucleotide sequence of gene DNA of the arginine deiminase mutant withimproved enzyme activity and temperature stability is shown as SEQ IDNo.2.

A recombinant plasmid comprises a DNA molecule.

A host cell comprises the DNA molecule or comprises the recombinantplasmid.

According to the arginine deiminase mutant with improved enzyme activityand temperature stability, an arginine deiminase mutant Gly292Pro withimproved enzyme activity and temperature stability was obtained bytransferring the recombinant plasmid comprising gene sequence DNA of anarginine deaminase mutant into an Escherichia coli BL21(DE3) host,constructing a mutant, and performing sequence verification andconfirmation. Glycine Gly at position 292 is mutated into proline Pro.

A construction method of the arginine deiminase mutant with improvedenzyme activity and temperature stability comprises the following steps:

1. designing a primer according to a gene sequence of arginine deiminasearcA of Enterococcus faecalis SK32.001; by taking Enterococcus faecalisSK32.001 comprising an arginine deiminase sequence as a template,obtaining a gene segment comprising arginine deiminase arcA by a PCRmethod to construct a recombinant plasmid with the connection to anexpression vector pET-28a;

2. using B-FITTER software to recognize key amino acid residues thathave adverse effects on temperature stability in enzyme molecules; thenusing SWISS-MODEL software to perform protein structure simulation onparent arginine deaminase, so as to obtain a tertiary structure ofarginine deaminase; through comparative analysis, determining an aminoacid site to be mutated is glycine at position 292;

3. designing a mutation primer, using a one-step PCR method to performsite-directed mutation on a nucleotide sequence of arginine deaminase,and replacing the amino acid at position 292 to obtain a recombinantvector comprising an arginine deaminase mutant gene sequence; and

4. enabling the recombinant vector comprising the arginine deaminasemutant gene sequence to enter competent cells of Escherichia coli E.coli BL21(DE3), inducing expression, collecting thalli, and using Ni-NTAfor protein separation and purification after ultrasonication on cellsto obtain an arginine deaminase mutant.

Application of the arginine deiminase mutant with improved enzymeactivity and temperature stability: the arginine deiminase mutant isused for citrulline production and medication.

Beneficial effects of the invention: the arginine deiminase mutantprovided by the invention has improved enzyme activity and temperaturestability, wherein the enzyme activity is increased by 1.5 times and thehalf-life period at 40° C. is increased by 5.43 times. The inventionoptimizes and improves relieves wild type arginine deiminase, solves theproblems of low enzyme activity and low temperature stability, andcreates favorable conditions for the use of the enzyme in citrullineproduction and medication.

The arginine deiminase gene arcA used in the invention is derived from astrain of Enterococcus faecalis that can produce citrulline, CCTCC NO: M2011465, which is deposited at the China Center for Type CultureCollection in Wuhan University, Wuhan, China, and is named as SK32.001(Enterococcus faecalis SK32.001), and has been published in Chinesepatent CN102433290A.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Construction map of recombinant plasmid pET-28a-ADIG292P

FIG. 2: Temperature stability of wild enzyme and mutant enzyme at 40° C.

FIG. 3: Conversion rate of biosynthesized citrulline of wild enzyme andmutant enzyme

DETAILED DESCRIPTION

The invention is further illustrated below by examples, which are forthe purpose of illustration and are not intended to limit the scope ofthe invention.

Materials and reagents: restriction enzyme, Solution I ligase, PCRreagents and the like used herein were all purchased from TaKaRa BioInc.; plasmid extraction kit, genome extraction kit, agarosepurification kit, escherichia coli DH5α, BL21 (DE3) strains and primerswere all purchased from Sangon Bioengineering (Shanghai) Co., Ltd.;other reagents were all analytical pure reagents purchased at home orabroad.

Example 1: Construction of Recombinant Plasmid

Enterococcus faecalis 5K32.001 was cultured to an exponential growthmetaphase, and 2 mL of a bacteria solution was centrifuged at 10000r/min for 10 min. Supernatant was discarded, and lysozyme treatment wasperformed for 30 min. Genomic DNA was extracted according to kitinstructions.

The following primers were designed for the amplification of arcA:

FADI-2: 5′-CGCGGATCCA TGAGTCATCC AATTAATGT-3′(containing BamH I restriction enzyme cutting sites), RADI-2:5′-CCGCTCGAGT TAAAGATCTT CACGGT-3′(containing Xho I restriction enzyme cutting sites).

PCR amplification conditions: 3 min of denaturation at 95° C., 30 cycles(95° C. 30 s, 55° C. 30 s, 72° C. 210 s), at last 2 min of extension at72° C.

After purification on an amplification product, a PCR product and avector pET-28a-c (+) were double-digested with BamH I and Xho I, and thedigested products were respectively recovered and joint with Solution Iligase for 2 h at 16° C. for heat shock transformation into DH5α cells.When transformants grew on a plate, single colonies were picked into aliquid medium, and the plasmid was extracted. A recombinant plasmidpET-28a-ADI was verified through enzyme digestion. The recombinantplasmid was transformed into BL21 (DE3) cells to obtain BL21(DE3)/pET-28a-ADI engineered bacteria.

Example 2: Determination of Arginine Deiminase Mutation Sites

Software B-FITTER was used to calculate a temperature factor (B-factor)of parent arginine deiminase amino acid residues to obtain amino acidresidues with the highest temperature factor in an enzyme molecule;SWISS-MODEL software was used to simulate an arginine deiminase proteinstructure to obtain a tertiary structure model of arginine deiminase;Discovery Studio software was used for analyzing a spatial structure ofand distance between the amino acid residues with the highesttemperature factor and an enzyme catalytic activity center; and an aminoacid site to be mutated was then determined to be glycine at position292.

Example 3: Site-Directed Mutation

The primer design was performed based on a coding gene encoding arcA inEnterococcus faecalis SK23.001.

G292P-forward primer: 5′-CATCCAGAAA TCGAACCTGG CTTGGTTGTTT T-3′, andG292P-reverse primer: 5′-TTCGATTTCT GGATGAATCG TAAATTTATC ATA-3′,

wherein an underlined part represents a codon corresponding to glycineat position 292, encoded by a mutant gene.

PCR Amplification System:

10 × Reaction Buffer 5 μL dNTP mix 1 μL Forward primer (100 ng/μL) 1.25μL Reverse primer (100 ng/μL) 1.25 μL Template pET-28a-ADI (10 ng) 2 μLpfuTurbo DNA polymerase 1 μL (2.5 U/μL) ddH₂O 38.5 μL

After PCR amplification, 1 μL of Dpn I restriction enzyme (10 U/μL) wasadded into the reaction solution and was thermally insulated at 37° C.for 1 hour to eliminate a template. A PCR product was transferred intoEscherichia coli DH5α cells to coat the plate. Single colonies werepicked to a liquid medium. A plasmid was extracted, and the correctmutant plasmid was obtained by sequencing. The successfully constructedmutant plasmid was transferred into Escherichia coli BL21 (DE3) toobtain a mutant strain BL21 (DE3)/pET-28a-ADIG292P.

Example 4: Expression and Purification of Wild Enzyme and Mutant Enzyme

Single colonies of BL21 (DE3)/pET-28a-ADI and BL21(DE3)/pET-28a-ADIG292P were picked and put in an LB culture mediumcontaining 0.5 mmol/L kanamycin, were cultured at 37° C. for 12 h at 200r/min, then were transferred to an LB culture medium containing 0.5mmol/L kanamycin, were cultured at 37° C. and 200 r/min until OD600 fellinto the range of 0.5 to 0.7, and 1 mmol/L IPTG was added at theconditions of 28° C. and 200 r/min for 9 h's induction.

After fermentation broth was centrifuged at 10000 r/min and 4° C. for 10min, supernatant was discarded. Then the fermentation broth was washedtwice with a phosphate buffer, a 15-20 mL phosphate buffer was added tosuspend thalli, and ultrasonication was performed for 15 min (power of22 W, 2 s of intermittence for each 1 s of ultrasonication).Centrifugation was performed at the conditions of 4° C. and 10000 r/minfor 10 min, and supernatant was collected as crude enzyme solution andwas filtered through a hydrophilic membrane with the pore diameter of0.22 μm.

A Binding Buffer was used to pre-balance Ni2+chelating agarose resincolumn; the crude enzyme solution was added, and then the Binding Bufferand Washing Buffer were used for balancing separately; an Elution Bufferwas used to elute the enzyme, and the enzyme was recovered; and therecovered enzyme solution was dialyzed in a dialysis buffer and then wasrestored in a 4° C. refrigerator.

Buffer Preparations Involved:

Phosphate Buffer (PB): 50 mmol/L, pH 5.5;

Binding Buffer: 50 mmol/L PB, 500 mmol/L NaCl, pH 7.0;

Washing Buffer: 50 mmol/L PB, 500 mmol/L NaCl, pH 7.0, 50 mmol/Limidazole;

Elution Buffer: 50 mmol/L PB, 500 mmol/L NaCl, pH 7.0, 500 mmol/Limidazole; and

the dialysis buffer: 50 mmol/L PB, pH 5.5, 10 mmol/L EDTA.

Example 5: Determination of Enzyme Activity and Temperature Stability ofWild Enzyme and Mutant Enzyme

Definition of enzyme activity: Under this condition, the amount ofenzyme for the catalytic production of 1 μmol of citrulline per minuteis defined as one enzyme activity unit (U).

Temperature stability: an enzyme solution was thermally insulated at 40°C., the time-gradient samples were taken out according to a timegradient, was added into a substrate L-arginine, and was placed in 45°C. water for a water bath for 10 min, and reaction was immediatelyterminated by boiling. Then citrulline yield was measured by HPLC, andrelative enzyme activity was calculated. The enzyme activity of theuntreated enzyme solution was defined as 100%, and the percentage ofrelative enzyme activity versus time was plotted to assess thetemperature stability of enzyme. Results obtained are shown in FIG. 2:half-life period of the enzyme of a mutant G292P was prolonged to 91.8min from 16.9 min of the wild enzyme, and was increased by 5.43 times.

Example 6: Efficient Synthesis of Citrulline

Separately take 1 g of bacteriophage of wild enzyme and mutant enzymeinto 50 mL L-arginine solution with a concentration of 100 g/L. Reactionwas carried out at 45, 150 r/min and pH 6.0-6.5, and timing sampling andcitrulline yield analysis were performed. Results are shown in FIG. 3:95% or more of arginine was converted into citrulline by wet thalli ofthe mutant enzyme after 2 h., and 95% or more of arginine was convertedinto citrulline by wet thalli of the wild enzyme after 5 h.

The citrulline yield in this example was 95% or more.

Through the invention, a higher concentration of citrulline can beobtained by enzymatic conversion in a relatively short period of time,which lays a foundation for future industrial application.

What is claimed is:
 1. An arginine deiminase mutant, comprising an aminoacid sequence set forth in SEQ ID NO:
 1. 2. A recombinant cell linecapable of expressing the arginine deiminase mutant of claim
 1. 3. Therecombinant cell line of claim 2, comprising fungi or bacteria cells. 4.An application of an arginine deiminase mutant, wherein the argininedeiminase mutant comprises an amino acid sequence set forth in SEQ IDNO:1, wherein the application comprises making an expression vector forexpressing the arginine deiminase mutant, and obtaining a protein formof the arginine deiminase mutant.
 5. The application of claim 4,comprising preparing a medicament for inhibiting arginine-deficienttumors, breast cancer or liver cancer cells.
 6. The application of claim4 comprising preparing a medicament for the treatment of leukemia. 7.The application of claim 4, comprising studying an antitumor activityand related pharmacological activities of medicines.
 8. The applicationof claim 4, comprising transferring the expression vector intoEscherichia coli BL21(DE3), and constructing a recombinant Escherichiacoli that express the arginine deiminase mutant.
 9. A nucleotidesequence of a gene encoding an arginine deaminase set forth in SEQ IDNO:
 2. 10. The application of claim 4, comprising using the argininedeiminase mutant in citrulline production.
 11. The application of claim10, comprising using the arginine deiminase mutant as biocatalyst incatalyzing arginine to produce citrulline.
 12. The application of claim4, comprising using cells that express the arginine deiminase mutant incatalyzing arginine to produce citrulline.
 13. The application of claim12, wherein the cells is a recombinant strain that express the argininedeiminase mutant.
 14. The application of claim 13, comprisingconstructing the recombinant strain by following steps: 1) designing aprimer according to a gene sequence of arginine deiminase arcA ofEnterococcus faecalis SK32.001; by taking Enterococcus faecalis SK32.001comprising an arginine deiminase sequence as a template, obtaining agene segment comprising arginine deiminase arcA by a PCR method toconstruct a recombinant plasmid with a connection to an expressionvector pET-28a; 2) using B-FITTER software to recognize key amino acidresidues that have adverse effects on temperature stability in enzymemolecules; then using SWISS-MODEL software to perform protein structuresimulation on parent arginine deaminase, so as to obtain a tertiarystructure of an arginine deaminase; through a comparative analysis,determining an amino acid site to be mutated is glycine at position 292;3) designing a mutation primer, using a one-step PCR method to performsite-directed mutation on a nucleotide sequence of arginine deaminase,and replacing the amino acid at position 292 to obtain a recombinantvector comprising an arginine deaminase mutant gene sequence; and 4)enabling the recombinant vector comprising the arginine deaminase mutantgene sequence to enter competent cells of Escherichia coli E. coliBL21(DE3), inducing expression, collecting thalli, and using Ni-NTA forprotein separation and purification after ultrasonication on cells toobtain the arginine deaminase mutant.